CHAPTER 3

Amino Acids, Peptides,

Proteins

Learning goals:
Structure and naming of amino acids
Structure and properties of peptides
Ionization behavior of amino acids and
peptides
Methods to characterize peptides and
proteins
 Proteins:
Main Agents of Biological
Function
Catalysis
enolase (in the glycolytic pathway)
DNA polymerase (in DNA replication)

Transport
hemoglobin (transports O2 in the blood)
lactose permease (transports lactose across the cell
membrane)

Structure
collagen (connective tissue)
keratin (hair, nails, feathers, horns)

Motion
myosin (muscle tissue)
actin (muscle tissue, cell motility)
Proteins serve a wide range of
biological functions
 Amino Acids:
Building Blocks of Protein
Proteins are linear heteropolymers of -amino
acids

Amino acids have properties that are well-suited

to carry out a variety of biological functions
Capacity to polymerize
Useful acid-base properties
Varied physical properties
Varied chemical functionality
 Amino acids share many
features, differing only at the
R substituent
 Most -amino acids are
chiral
The -carbon always has four
substituents and is tetrahedral
All (except proline) have:
an acidic carboxyl group
a basic amino group
an -hydrogen connected to the -carbon
The fourth substituent (R) is unique
In glycine, the fourth substituent is also
hydrogen
Amino Acids: Atom Naming
Organic nomenclature: start from one end
Biochemical designation:
start from -carbon and go down the R-group
All amino acids are chiral
(except glycine)
Proteins only contain L amino acids
 Amino Acids:
Classification
Common amino acids can be placed in five
basic groups depending on their R
substituents:
Nonpolar, aliphatic (7)
Aromatic (3)
Polar, uncharged (5)
Positively charged (3)
Negatively charged (2)
These amino acid side chains absorb UV light at 270280
nm
These amino acids side chains can form hydrogen
bonds.
 Uncommon Amino Acids in
Proteins
Not incorporated by ribosomes
except for Selenocysteine
Arise by post-translational modifications
of proteins
Reversible modifications, especially
phosphorylation, are important in
regulation and signaling
Modified Amino Acids Found in
Proteins
Reversible Modifications of
Amino Acids
 Ionization of Amino Acids

At acidic pH, the carboxyl group is protonated

and the amino acid is in the cationic form.
At neutral pH, the carboxyl group is
deprotonated but the amino group is
protonated. The net charge is zero; such ions
are called Zwitterions.
At alkaline pH, the amino group is neutral
NH2 and the amino acid is in the anionic form.
Cation Zwitterion
Anion
Chemical Environment Affects pKa Values

-carboxy group is much more acidic than in

carboxylic acids
-amino group is slightly less basic than in
amines
 Amino acids can act as
buffers
Amino acids with uncharged side chains, such
as glycine, have two pKa values:
The pKa of the -carboxyl group is 2.34
The pKa of the -amino group is 9.6

It can act as a buffer in two pH regimes.

Buffer
Regio
ns
Amino acids carry a net charge
of zero
at a specific pH (the pI)
Zwitterions predominate at pH values between the
pKa values of the amino and carboxyl groups
For amino acids without ionizable side chains, the
Isoelectric Point (equivalence point, pI) is
pK1 pK 2
pI
2

At this point, the net charge is zero

AA is least soluble in water
AA does not migrate in electric field
 Ionizable side chains can
show up in titration
curves
Ionizable side chains can be also titrated
Titration curves are now more complex
pKa values are discernable if two pKa
values are more than two pH units apart
 How to Calculate the pI
When the Side Chain is
Ionizable
Identify species that carries a net zero
charge
Identify pKa value that defines the acid
strength of this zwitterion: (pK2)
Identify pKa value that defines the base
strength of this zwitterion: (pK1)
Take the average of these two pKa values
 Formation of Peptides
Peptides are small condensation products of amino acids
They are small compared to proteins (M w < 10 kDa)

<100AAs,
typicallymuch
less

This is not really

how it works but
a net reaction!
 Peptide ends are not the same
Numbering (and naming) starts from the amino
terminus
AA1 AA2 AA3 AA4 AA5
 Naming peptides:
start at the N-terminus
O O O O O O O
+
H3N CH C N CH C N CH C N CH C N CH C N CH C N CH C O-
H H H H H H
CH2 CH2 CH2 CH2 CH CH3 CH2 CH OH

SH CH2 CH2 CH2 OH CH3

N
C O S CH3
NH
OH CH3

Using full amino acid names

cysteinylhistadylglutaminylmethionylisoleucylsery
lthreonine
Using the three-letter code abbreviation
cys-his-glu-met-ile-ser-threonine
For longer peptides (like proteins) the one- letter
code can be used
CHEMIST
 Peptides: A Variety of
Functions
Hormones and pheromones
insulin (think sugar)
oxytocin (think childbirth)
sex-peptide (think fruit fly mating)

Neuropeptides
substance P (pain mediator)

Antibiotics
polymyxin B (for Gram bacteria)
bacitracin (for Gram + bacteria)

Protection, e.g., toxins

amanitin (mushrooms)
conotoxin (cone snails)
chlorotoxin (scorpions)
 Proteins, typically > 100
AAs
Polypeptides (covalently linked -amino acids) + poss
cofactors
functional non-amino acid component
metal ions or organic molecules
coenzymes
organic cofactors
NAD+ in lactate dehydrogenase
prosthetic groups
covalently attached cofactors
heme in myoglobin
other modifications
Polypeptide size and number
varies greatly in proteins
Classes of Conjugated
Proteins
 What to Study about
Peptides and Proteins

What is its sequence and composition?

What is its three-dimensional structure?
How does it find its native fold?
How does it achieve its biochemical role?
How is its function regulated?
How does it interacts with other
macromolecules?
How is it related to other proteins?
Where is it localized within the cell?
What are its physico-chemical properties?
A mixture of proteins can be
separated
Separation relies on differences in
physical and chemical properties
Charge
Size
Affinity for a ligand
Solubility
Hydrophobicity
Thermal stability
Chromatography is commonly
used for preparative separation
Column Chromatography

In lecture we will
consider what the
chromatograms look
like and elution
techniques
Separation by Charge
Separation by Size
Separation by Affinity
 Affinity Tech
Although many proteins have structural features that
lend themselves to affinity purification,
The chromatographic media on which they could be
purified is often $$

Further, some proteins do not lend themselves to affinity

purification (no recognition site and binding partner)

Two solutions:
1.Immunoaffinity: make antibody to protein, immobilize
antibody
2.Use recombinant DNA tech to place a sequence of
amino acids at N- or C- terminus of protein that can be
used with an affinity reagent
Electrophoresis for Protein
Analysis
Separation in analytical scale is
commonly done by electrophoresis
Electric field pulls proteins according to
their charge
Gel matrix hinders mobility of proteins
according to their size and shape

Typically,thisisrunasadenaturingtechniqueandisthus
notsuitedforobtainingproteinsintheirbiologicallyactive
form
 SDS PAGE: Molecular
Weight
SDS sodium dodecyl sulfate a detergent

SDS micelles bind to and unfold all the

proteins
SDS gives all proteins an uniformly negative
charge
The native shape of proteins does not matter
Rate of movement will only depend on size:
small proteins will move faster
 SDS-PAGE can be used to
calculate the molecular weight
of a protein
 Isoelectric focusing can be
used to determine the pI of a
protein
Isoelectric focusing and SDS-
PAGE are combined in 2D
electrophoresis
Spectroscopic Detection of
Aromatic Amino Acids
The aromatic amino acids absorb light in the
UV region
Proteins typically have UV absorbance
maxima around 275280 nm
Tryptophan and tyrosine are the strongest
chromophores
Concentration can be determined by UV-
visible spectrophotometry using Beers law:
A = cl
Specific activity (activity/total
protein) can be used to assess
protein purity
 Protein Sequencing
It is essential to further biochemical analysis that we
know the sequence of the protein we are studying
Actual sequence generally determined from DNA
sequence
Edman Degradation (Classical method)
Successive rounds of N-terminal modification, cleavage,
and identification
Can be used to identify protein with known sequence
Mass Spectrometry (Modern method)
MALDI MS and ESI MS can precisely identify the mass of a
peptide, and thus the amino acid sequence
Can be used to determine post-translational modifications
Edmans Degradation
MS Procedures for Sequence IDs
 Protein Sequences as Clues to Evolutionary
Relationships

Sequences of homologous proteins from a

wide range of species can be aligned and
analyzed for differences

Differences indicate evolutionary divergences

Analysis of multiple protein families can

indicate evolutionary relationships between
organisms, ultimately the history of life on
Earth
 Chapter 3: Summary

In this chapter, we learned about:

The many biological functions of peptides

and proteins
The structures and names of amino acids
found in proteins
The ionization properties of amino acids and
peptides
The methods for separation and analysis of
proteins